Anthocyanin content and profile within and among blueberry species

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1 Anthocyanin content and profile within and among blueberry species W. Kalt 1, J. E. McDonald, R. D. Ricker 2, and X. Lu 3 Agriculture and Agri-Food Canada, 1 Atlantic Food and Horticulture Research Centre, 32 Main Street, Kentville, Nova Scotia, Canada B4N 1J5 ( kaltw@em.agr.ca). 2 Hewlett Packard Company, Little Falls Analytical Division-Newport, 538 First State Blvd., Newport, DE, USA, ; and 3 Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 43 McGillvray Street, c/o University of Guelph, Guelph, Ontario, Canada N1G 2W1. Received 2 February 1999, accepted 9 April Kalt, W., McDonald, J. E., Ricker, R. D. and Lu, X Anthocyanin content and profile within and among blueberry species. Can. J. Plant Sci. 79: Anthocyanins in ripe fruit of four Vaccinium species and genotypes within these species were compared, revealing substantial inter- and intra-species variability among these commercial and non-commercial blueberries. The highest total anthocyanin content occurred in the bilberry (V. myrtillus L.). Commercial lowbush blueberry clonal mixtures and wild velvet leaf blueberries (V. myrtilloides L.) had about 43% of the anthocyanin content of bilberries (fresh weight basis). Three commercial highbush cultivars (V. corymbosum L.) had about 30 % of the bilberry level, while wild genotypes of highbush blueberries had almost 60% of the bilberry level. Acetylation of anthocyanins occurred widely among all species, except bilberries. Although the proportions of the five blueberry anthocyanidins varied substantially among the commercial blueberries, these differences probably do not contribute substantially to differences in their relative antioxidant capacity. Key words: Vaccinium, bilberry, acetylation, cluster analysis, antioxidant Kalt, W., McDonald, J. E., Ricker, R. D. et Lu, X Teneur en anthocyanine et variabilité intra et interspécifique chez certaines espèces de Vaccinium. Can. J. Plant Sci. 79: Dans une étude comparée de la teneur en anthocyanine du fruit mûr de quatre espèces de Vaccinium et de divers génotypes dans chacune d elles, nous avons relevé une variabilité importante, tant dans une même espèce qu entre les espèces, qu il s agisse ou non d espèces exploitées commercialement. La plus haute teneur en anthocyane a été observée chez la myrtille vraie (V. myrtillus L.). Les mélanges clonaux de bleuet nain d exploitation commerciale et l airelle fausse-myrtille (V. myrtilloïdes L.) contenaient environ 43 % de la teneur mesurée dans les myrtilles vraies (calculé sur le poids du fruit frais). Chez les trois cultivars commerciaux comparés de bleuet en corymbes (V. corymbosum L.), cette proportion par rapport à la myrtille n était que de 30 %, mais elle remontait à 60 % dans trois clones sauvages. L acétylation des anthocyanines était fréquente parmi toutes les espèces, sauf la myrtille vraie. Bien que les proportions des cinq anthocyanidines du bleuet variaient de façon importante parmi les bleuets du commerce, ces différences ne signifiaient probablement pas grand-chose pour le pouvoir antioxydant relatif des fruits. Mots clés: Vaccinium, myrtille, acétylation, analyse hiérarchique, antioxydant North American blueberry species of commercial importance include highbush blueberry (V. corymbosum L.), lowbush blueberry (V. angustifolium Aiton), and rabbiteye (V. ashei Reade). Highbush and lowbush blueberries represent approximately equal proportions of North American blueberry production, while relatively small quantities of rabbiteye fruit are limited to markets in the Southeastern United States. The velvet leaf blueberry (V. myrtilloides L.) is found at up to 50% abundance in stands of lowbush blueberries in some production regions (S. Van der Kloet, personal communication). In Europe, the native commercial blueberry industry is based on the bilberry or European blueberry (V. myrtillus L.). A feature that distinguishes highbush and lowbush blueberries is that highbush blueberries have been improved through artificial selection and are grown as unique varieties, while lowbush blueberries grow wild. Commercial lowbush blueberries are a highly heterogenous mixture of wild genotypes of V. angustifolium, containing smaller amounts of velvet leaf blueberry. Like lowbush blueberries, 617 European bilberries grow wild and the commercial product is a mixture of genotypes. Compared with many small fruits, blueberries have a high concentration of anthocyanin pigments. Bilberries belong to the Vaccinium subgenera Myrtillus, which contain anthocyanins in both their peel and flesh (Ballington et al. 1987). Highbush, lowbush, rabbiteye and velvet leaf blueberries, members of the subgenera Cyanococcus, have anthocyanin only in the peel. Blueberries contain five of the six anthocyanidins commonly found in nature (Mazza and Miniati 1993). As is the case for all anthocyanidins commonly found in nature (Mazza and Miniati 1993). As is the case for all anthocyanidins, they occur in blueberry fruit as glycosides (anthocyanidin glycoside = anthocyanin), and may have acyl groups attached to the sugar moiety (Gao and Mazza 1994). Compared with other fruit, lowbush blueberries have a complex mixture of anthocyanins (Mazza and Miniati 1993). Anthocyanin stability in processed products has been studied and certain acylated anthocyanins have been shown to be particularly stable after extraction (Dougall et al. 1997).

2 618 CANADIAN JOURNAL OF PLANT SCIENCE Table 1. Total anthocyanin content (mg g 1 fresh weight) among blueberry species and genotypes. Values are the mean of triplicate samples Species Common name Genotype mg g 1 FW z % of bilberry V. myrtillus European bilberry Mixture V. angustifolium Lowbush blueberry Mixture Region V. angustifolium Lowbush blueberry Mixture Region V. angustifolium Lowbush blueberry Mixture Region V. angustifolium Lowbush blueberry Blomidon V. angustifolium Lowbush blueberry Cumberland V. angustifolium Lowbush blueberry Fundy V. corymbosum Highbush blueberry Bluecrop V. corymbosum Highbush blueberry Coville V. corymbosum Highbush blueberry Jersey V. corymbosum Highbush blueberry 1 (wild) V. corymbosum Highbush blueberry 2 (wild) V. corymbosum Highbush blueberry 3 (wild) V. myrtilloides Velvet leaf blueberry 1 (wild) V. myrtilloides Velvet leaf blueberry 2 (wild) V. myrtilloides Velvet leaf blueberry 3 (wild) z Fresh weight basis, calculated using the extinction coefficient for malvidin 3-glucoside. Significant differences: Species P < 0.001; Clones within species P < Specific comparisons: Highbush vs. bilberry, lowbush and velvet leaf blueberries P < 0.001; Cultivated highbush vs. wild highbush P < Anthocyanins are antioxidants (Wang et al. 1997) and dietary antioxidants are believed to play a role in reducing the risks of various human degenerative diseases (Prior and Cao 1998). Since blueberries contain a comparitively high level of anthocyanins, there may be potential value-added opportunities for blueberries as food supplements. Anthocyanins from the European bilberry have been well studies with regard to human health (Morazzoni and Bombardelli 1996), and bilberry anthocyanin extract is currently marketed in a variety of pharmaceutical and food supplement products (Kalt and Dufour 1997). In this study, the diversity in anthocyanins among Vaccinium species was explored in commercial and noncommercial blueberries. Where possible, single genotypes and mixtures of genotypes are compared within a species. Results are discussed with respect to inter- and intra-species differences in simple and acylated anthocyanins, and relative antioxidant capacities of these species. MATERIALS AND METHODS Samples Clonal mixtures of European bilberries from different regions of Europe were obtained from D. Dufour, Ferlux- Mediolanum, Clermont-Ferrand, France. Ripe fruit of wild lowbush blueberries were harvested from three regions of Nova Scotia; samples included at least 20 clones. At least three subsamples of bilberry and lowbush blueberry were analyzed. Three replicate samples of ripe fruit of the lowbush clones Blomidon, Cumberland and Fundy, propagated from wild selections, were obtained from the Agriculture and Agri-Food Canada (AAFC) Sheffield Farm, Nova Scotia, Canada. Highbush blueberry varieties Bluecrop, Coville and Jersey were each collected from three separate shrubs from Blueberry Acres, Sheffield Mills, Nova Scotia. Replicate samples of ripe fruit from three genotypes of wild highbush blueberries were harvested from a planting at the AAFC Research Centre, Kentville, Nova Scotia. Three clones of the velvet leaf blueberry were harvested from the wild, and three subsamples used for analysis. Fruit was stored at 70 C until analysis. Sample Preparation Frozen fruits were shredded in a food processor without thawing. Approximately 10 g of the mix was homogenized for 6 min in a Virtis Homogenizer (Gardiner, NY) with 10 ml of extraction solvent of MeOH/Formic acid/water (70/2/28, M/F/W) and 2.5 g of Celite filtering aid. After grinding and coarse filtering, the homogenate was left for 60 min to equilibrate then filtered through a 0.45-µM Duropore filter (Millipore, Canada) prior to injection onto the HPLC. HPLC Analysis A 50-µL injection of filtrate was separated on a SB-C18 column (5 µm, 250 mm 4.6 mm; 4 10 mm guard column; Zorbax, Hewlett-Packard Canada, Mississauga, ON) using a Beckman System Gold HPLC (Palo Alto, CA) equipped with a Model 126 binary pump system and a Model 167 Scanning UV Detector. Data were collected at 280 and 520 nm. Compounds on the column were separated with 5% formic acid in water (solvent A) and methanol (solvent B), using a gradient elution program; 0 10 min, 14 17% B; min, 17 23% B; min, 23 47% B; min, 47 14% B; min 14% B. Flow rate was 1.5 ml min 1. Total anthocyanin content of the samples was determined by the spectrophotometric method of Wrolstad (1976), and data were calculated using the molar extinction coefficient for malvidin 3-glucoside (28 000). HPLC and spectrophotometric data were analyzed using Genstat (Genstat Committee 1993). Hierarchical Cluster Analysis Sample means of normalized peak areas of each chromatogram were calculated for each genotype. There were total of 16 genotypes with 25 peaks for each genotype. The data were analyzed with the HCLUSTER directive in Genstat 5 Release 3 (1993), where means of normalized

3 KALT ET AL. BLUEBERRY ANTHOCYANINS 619 Fig. 1. HPLC chromatograms of anthocyanins (A 520 ) from extracts of blueberry species. Panel A. Lowbush blueberry Fundy; B, bilberry; C. Velvet leaf blueberry; D. Highbush blueberry Coville. Note: Coville was loaded at twice the volume of samples in Panels A C. Table 2A. Anthocyanin content z of individual clones and clonal mixtures of wild lowbush blueberry Anthocyanin Lowbush Lowbush Lowbush Lowbush Lowbush Lowbush peak y Blomidon Cumberland Fundy Mixture 1 Mixture 2 Mixture 3 Peak area g 1 fresh weight ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0 4 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.65 z Anthocyanin content expressed as peak area per gram fresh weight; total peak area normalized to y Peak 1, delphinidin 3-galactoside; 2, delphinidin 3-glucoside; 3, cyanidin 3-galactoside; 4, delphinidin 3-arabinoside; 5, cyanidin 3-glucoside; 6, petunidin 3-galactoside; 7, cyanidin 3-arabinoside; 8, petunidin 3-glucoside; 9, peonidin 3-galactoside; 10, petunidin 3-arabinoside; 11, peonidin 3-glucoside; 12, malvidin 3-galactoside; 13, delphinidin 3-galactoside acetylated; 14, malvidin 3-glucoside; 15, malvidin 3-arabinoside; 16, cyanidin 3-galactoside acetylated; 17, cyanidin 3-arabinoside acetylated; 18, delphinidin 3-glucoside acetylated; 19; petunidin 3-galactoside acetylated; 20, peonidin 3-galactoside acetylated; 21, cyanidin 3-glucoside acetylated; 22, malvidin 3-galactoside acetylated; 23, petunidin 3-glucoside acetylated; 24, peonidin 3-glucoside acetylated; 25, malvidin 3-glucoside acetylated.

4 620 CANADIAN JOURNAL OF PLANT SCIENCE Table 2B. Anthocyanin content z of velvetleaf blueberry and bilberry Anthocyanin Velvet leaf Velvet leaf Velvet leaf peak y blueberry1 blueberry2 blueberry3 Bilberry Peak area g 1 fresh weight ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.22 z Anthocyanin content expressed as peak area per gram fresh weight; total peak area normalized to y Peak identification as in Table 2A. peak areas for the samples were used to calculate the degree of similarity among the genotypes, according to the Euclidean test type. HCLUSTER directive provides hierarchical cluster analysis. The aim of cluster analysis is to arrange the 16 genotypes into more or less homogeneous groups. A dendrogram was used to display the tree fitted to the similarity matrix and provide a scale to show the level similarity at which the fusions occurred. Eighty percent was specified as the threshold value to form groups from the dendrogram. RESULTS AND DISCUSSION Total anthocyanin content varied among the Vaccinium species and genotypes within a species (Table 1). Bilberry anthocyanins are compared with the other blueberry samples, since its anthocyanin content is the highest among the samples. Also bilberries are the Vaccinium best recognized for their health functionality. The commercial lowbush blueberry clonal mixtures had about 40% of the level of anthocyanins as the bilberry (fresh weight basis). The individual lowbush clones Blomidon, Cumberland and Fundy were different in their anthocyanin content, ranging between 26 and 69% of the bilberry level. The relative ranking in pigment content among these three lowbush genotypes was the same as those reported by Kalt and McDonald (1996), although the absolute amounts were different. This suggests the influence of both genetic and environmental factors on blueberry anthocyanin content. The anthocyanin content of the commercial highbush cultivars Bluecrop, Coville and Jersey was about 30% of the bilberry level. The three wild highbush genotypes ranged between 45 and 68% of the anthocyanin level of bilberry. The relatively high anthocyanin content of the wild highbush genotypes was probably due to their small fruit size. The velvet leaf blueberries were similar to the lowbush blueberries in their total anthocyanin content, with relatively little variation among the three genotypes. Chromatograms of selected samples among the four Vaccinium species illustrate the qualitative and quantitative variation in individual anthocyanin profiles (Fig. 1). The anthocyanins of the lowbush clone Fundy were identified by Gao and Mazza (1995) and confirmed in the present study using electrospray LC-MS, with analysis of the base peak chromatograms of individual peaks (data not shown). The identity of compounds was verified, except in cases where masses were identical, for example, for the galactoside and glucoside of a given anthocyanidin. The HPLC profile of the lowbush clone Fundy (Fig. 1A) was virtually identical to that of Gao and Mazza (1995) except that the elution order of peaks 13 and 14 were reversed. In both studies, peaks 13 and 14 were not well separated. Gao and Mazza (1995) identified the smaller of the two peaks as delphinidin 3- galactoside acetylated, and the larger peak as malvidin 3- glucoside. These results were confirmed by LC-MS in the present study. The Fundy anthocyanin profile was used for peak identification, since it contained all of the 25 peaks observed among the samples in the study. Sample means of the normalized peak areas of chromatograms are reported in Tables 2A-C and were used to calculate statistical similarities for Fig. 2. Peaks 1-12 and peak 14 and 15 are monosides of five different anthocyani-

5 KALT ET AL. BLUEBERRY ANTHOCYANINS 621 Table 2C. Anthocyanin content z in cultivated and wild highbush blueberries Anthocyanin Highbush Highbush Highbush Wild Wild Wild peak y Bluecrop Coville Jersey Highbush 1 Highbush 2 Highbush 3 Peak area g 1 fresh weight ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.58 z Anthocyanin content expressed as peak area per gram fresh weight; total peak area normalized to y Peak identification as in Table 2A. dins. Peak 13 and peaks are anthocyanidin glycosides, which are acylated with acetic acid groups. The mixtures of bilberry clones contained three different glycosides of five anthocyanidins, and contained essentially no acylated anthocyanins (Fig. 1B, Table 2B). In contrast to bilberry, the lowbush clone Fundy, and the clonal mixtures of wild lowbush blueberries contained acylated anthocyanins (Table 2A). Only acetylated anthocyanins (i.e., acetyl groups linked to either glucose, galactose or arabinose) were identified by Gao and Mazza (1995) and observed in the present study. Fundy exceeded the wild lowbush clonal mixtures in its relative amount of acetylated anthocyanins. The lowbush clones Blomidon and Cumberland contained no acetylated anthocyanins (data not shown), confirming the results of Gao and Mazza (1994). Since acetylated anthocyanins appear in some, but not all lowbush clones, intermediate levels of acetylated anthocyanins may be expected, and were indeed observed, when many clones were pooled as in the commercial product (Table 2A). This result is reflected in the dendrogram (Fig. 2), which summarizes results of hierarchical cluster analysis and places various blueberry samples in groups based on their percent of similarity. All of the lowbush samples occurred in the same group, with about 90% similarity. At a dissimilarity of about 10%, the Blomidon and Cumberland lowbush clones were separated from Fundy and the clonal mixtures, due to their lack of acylation. Although the cultivated and wild highbush blueberry genotypes differed substantially in their total anthocyanin content, their proportions of anthocyanins had a relatively high degree of similarity (approximately 85%) (Table 2C, Fig. 2). An exception was the wild genotype 2, which was markedly different from both the wild and cultivated highbush samples in more than half of its peaks (i.e. peaks 1 8, 10, 12, 15, and 25) (Table 2). The wild highbush genotype 2 was more similar to bilberry and the velvet leaf blueberry genotype 1, than to any of the highbush samples (Fig. 2). Acetylation in highbush blueberries was noted only recently by Gao and Mazza (1994), in the cultivar Bluecrop. Acetylation was also found among the wild highbush genotypes. Among the three wild highbush genotypes, the proportion of anthocyanins that were acetylated was greater than, approximately equal to, and less than that observed in the highbush cultivar Bluecrop (Table 2C). The anthocyanins of the velvet leaf blueberry, which have not been well studied, were most similar to the lowbush blueberry, and contained substantial amounts of acetylated anthocyanins (Table 2A and B). Among the three velvet leaf blueberry genotypes, the first was substantially different from the other two, particularly in the relative amounts of peaks 2, 8, 14, 15, 22 and 25 (Table 2B). These differences were so great that velvet leaf genotype 1 was segregated from the other two genotypes, between the two largest (i.e. least similar) groupings of the dendrogram (Fig. 2). The velvet leaf genotype 1 was grouped with the bilberry, and a wild highbush genotype. The potential importance of anthocyanin flavonoids as a source of dietary antioxidants has sparked interest in the relative antioxidant capacity of various anthocyanidin glycosides, and their content in fruits. Wang et al. (1997) have

6 622 CANADIAN JOURNAL OF PLANT SCIENCE Fig. 2. Dendrogram illustrating percent similarity among anthocyanins of blueberry samples, based on a normalized mean total peak area. used the oxygen radical absorbing capacity (ORAC) assay (Cao et al. 1995) to measure the antioxidant capacity of five of the six common anthocyanidins against the peroxyl radical, which is a common free radical in the body. The values are cyanidin = 2.24; malvidin = 2.01; delphinidin = 1.81; peonidin = 1.69; pelargonidin = 1.54 µmol Trolox equivalents µmol 1. (Trolox is a water-soluble vitamin E analogue.) No ORAC value for petunidin is reported, although based on its substitution pattern, it may have a value at least as high as peonidin (see discussion in Wang et al. 1997). Although the bilberry, lowbush and highbush blueberry had substantially different proportions of anthocyanidins (Table 3), their ORAC level would differ from each other by no more than 6%, based on a standardized level of anthocyanidin. With respect to their anthocyanin profiles alone, these species would be equally suitable sources of anthocyanin antioxidants. The antioxidant capacity (as ORAC) of the whole fruit extracts of blueberry species shown in Table 3 have been reported (Prior et al. 1998) with bilberry > lowbush blueberry > highbush blueberry. Thus, the total anthocyanin content, and not the anthocyanin profile is likely the major determinant of ORAC among blueberry species. In summary, results of the present study illustrate substantial inter- and intra-species variability in the total amount and relative proportions of anthocyanins among blueberries. Velvet leaf blueberries, which can occur in substantial abundance in stands of lowbush blueberries, are quite similar to lowbush blueberry in their anthocyanin content and profile. Acetylation of anthocyanins appeared to be quite widespread among highbush, lowbush and velvet leaf Table 3. Percent contribution of anthocyanidins among commercially important blueberry species Lowbush Highbush Anthocyanidin Bilberry blueberry z blueberry y % of total anthocyanidins Cyanidin Delphinidin Malvidin Peonidin Petunidin z Mix of wild clones. y Mean value of cultivars Bluecrop, Coville and Jersey. blueberries, although it did not occur in all genotypes. Acetylation was not observed in bilberries. The relative proportion of different anthocyanidins is probably not a major factor influencing the contribution that these pigments make to the total antioxidant capacity of blueberry fruit. Ballington, J. R., Ballinger, W. E., Maness, E. P. and Luby, J. J Anthocyanin, aglycone, and aglycone sugar content in the fruits of five species of Vaccinium section Myrtillus. Can. J. Plant Sci. 68: 241. Cao, G., Verdon, C. P. Wu, A. H. B., Wang, H. and Prior, R. L Automated oxygen radical absorbing capacity assay using the COBAS FARA II. Clinical Chem. 41: Dougall, D. K., Baker, D. C., Gakh, E. and Redus, M Biosynthesis and stability of monoacylated anthocyanins. Food Technol. 51: Gao, L. and Mazza, G Quantitation and distribution of simple and acylated anthocyanins and other phenolics in blueber-

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